In stroke, cerebral blood vessels contract or become so occluded that blood flow is blocked. The affected brain tissues suffer from a transient event of lack of oxygen, or ischemia, which leads to neuronal injury and the activation of neuronal plasticity mechanisms that limit the extent of the damage. However, activation of astrocytes may enhance the extent of tissue damage in the brain following an ischemic insult. After mechanical or ischemic trauma to the central nervous system, the release of nucleotides together with neurotransmitters into the extracellular space activate P2Y nucleotide receptors and can act in synergistic combination with growth factors to stimulate astrocyte proliferation. While much is known about the factors that cause the activation of astrocytes, very little is known about the molecular mechanisms that lead to astrocyte activation during ischemic events. To understand these mechanisms and the role of extracellular nucleotides, it will be important to elucidate the molecular determinants by which P2Y2 receptors activate signal transduction pathways in astrocytes. Therefore, we propose to characterize the signal transduction pathways coupled to P2Y2 receptors that mediate astrocyte activation using primary astrocytes from neonatal rodent brain, immortalized astrocytes (DITNC cells) and normal human astrocytes. We will test the hypothesis that an Arg-Gly-Asp motif in the first extracellular loop of the P2Y2 receptor promotes efficient coupling hypothesis that an Arg-Gly-Asp motif in the first extracellular loop of the P2Y2 receptor promotes efficient coupling to PLC that is dependent upon receptor association with the alphavbeta3/beta5 integrins. The relevance of P2Y2 receptor/integrin interactions in astrocytes to the activation of intracellular MAPK cascades also will be investigated. Furthermore, we will test the hypothesis that P2Y2 receptor agonists cause trans-activation of the tyrosine kinase activity of the EGF receptor and that this interaction plays a role in the physiological outcomes of P2Y2 receptor activation in astrocytes. Finally, we will probe the hypothesis that cerebral ischemia up-regulates P2Y2 receptor death. These studies will utilize the rat model of focal ischemia induced by occlusion of the middle cerebral artery (MCA) and the model of global ischemia induced by ligation of he common carotid arteries (CCA). The studies proposed here will provide novel targets for the development of therapeutic strategies for the management of brain injury after stroke, synaptogenesis, as well as other neurological conditions.